Francisella tularensis, a facultative intracellular gram-negative coccobacillus, is the etiological agent of tularemia, which can be a fatal disease in humans. One of the striking features of tularemia is the very low infectious dose and the high mortality rate associated with the pneumonic form of the disease usually acquired through aerosols. Taken together, these features make F. tularensis an attractive organism to be used in biological warfare, leading NIAID to include this organism as a class A bioterrorism agent. Very little is known about the pathogenesis mechanisms of F. tularensis, and the goal of this grant proposal is to identify F. tularensis Schu4 virulence genes. A better understanding of F. tularensis pathogenesis is essential for the design and development of a suitable vaccine. Bacterial pathogens express their virulence genes in concert within the host. Specific Aim 1 is designed to identify F. tularensis genes activated within macrophages using gene microarrays and differential fluorescence induction. In Specific Aim 2 we shall identify genes essential for disease in mice using signature tagged mutagenesis. The potential virulence genes identified through Specific Aims 1 and 2 will have their role in virulence directly assessed by generation of isogenic mutants and complemented strains. Therefore in Specific Aim 3 we will develop genetic systems to study the role of candidate F. tularensis Schu4 virulence genes. These combined studies will be germane to the identification of novel vaccine candidates and markers for F. tularensis detection.